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Abstract

The intracellular mobility of biomolecules is determined by transport and diffusion as well as molecular interactions and is crucial for many processes in living cells. Methods of fluorescence microscopy like confocal laser scanning microscopy (CLSM) can be used to characterize the intracellular distribution of fluorescently labeled biomolecules. Fluorescence correlation spectroscopy (FCS) is used to describe diffusion, transport and photo-physical processes quantitatively. As an alternative to FCS, spatially resolved measurements of mobilities can be implemented using a CLSM by utilizing the spatio-temporal information inscribed into the image by the scan process, referred to as raster image correlation spectroscopy (RICS). Here we present and discuss an extended approach, multiple scan speed image correlation spectroscopy (msICS), which benefits from the advantages of RICS, i.e. the use of widely available instrumentation and the extraction of spatially resolved mobility information, without the need of a priori knowledge of diffusion properties. In addition, msICS covers a broad dynamic range, generates correlation data comparable to FCS measurements, and allows to derive two-dimensional maps of diffusion coefficients. We show the applicability of msICS to fluorophores in solution and to free EGFP in living cells.

Figures (6)

Principle of RICS and msICS. (a) Confocal images scanned unidirectionally with well defined scanning velocity, pixel size, pixel dwell time and line scan time are subject to a two-dimensional spatial autocorrelation analysis. (b) For RICS, a two-dimensional autocorrelation function for a given scanning velocity is generated and fitted with a model function describing transport/diffusion, photophysical and interaction process to extract the spatio-temporal information inscribed into the image by the scanning process. (c) For msICS, a one-dimensional representation of the resulting autocorrelation function for a given pixel shift is generated as a function of the lag time given by the pixel dwell time for smaller and by the line time for larger times resulting from different scanning velocities and is fitted accordingly.

Characterization of the PSF. (a) Mean fluorescence intensity of Alexa488 taken from confocal images (circles, left axis) and the fit of Eq. (7) (red line) as well as the focal radius normalized to the value determined at 30 μW (squares, right axis) as a function of incident laser intensity. The vertical line marks the saturation power. (b) Map of the focal radius w0 and (c) of the focal volume V0 as determined with FCS of Alexa488 in solution on a 9 × 9 array of points covering an area of 243 × 243 μm2. The variations are very small within the central 135 × 135 μm2 area where the experiments were carried out.

msICS in solution. (a) msICS, ξ=2, of Alexa488 (20 nM) from a ROI of 200×200 pixels and 6×6 μm2 (circles) and fit (line) with Eq. (2). (b) msICS, ξ=1…10 (circles, decreasing amplitude with increasing ξ), of the same sample and ROI with global fit (lines). (c) Point FCS of the same sample (circles) and fit (line) with Eq. (7). For fit results see Table 2.